This invention relates to a process which increases the catalytic capacity of alcohol oxidases (EC 1.3.13). These enzymes occur in a number of micro-organisms, acting to convert a lower alkyl alcohol, such as methanol or ethanol, to a lower alkyl aldehyde, such as formaldehyde or acetaldehyde and hydrogen peroxide in the presence of oxygen. The use of alcohol oxidase enzymes to manufacture commercially important chemicals from inexpensive feedstocks provides an alternative which could be economically superior to the synthetic processes now in use.
An enzyme suitable for commercial processes should be cheap and productive. An organism able to use an inexpensive feedstock and in which the alcohol oxidase can be induced to very high levels would obviously provide an economical source of enzyme. Such an enzyme must further satisfy productivity criteria. Specifically, it must tolerate high concentrations of substrates and products and have a high catalytic capacity, an ability to convert a relatively high amount of substrate to products before inactivation. Catalytic capacity is defined as the ratio of two rate constants, the first determined by the rate at which the enzyme converts substrate into product, having a constant, K.sub.cat, and the second determined by the rate at which the enzyme becomes inactivated by product, having a constant, K.sub.inact : ##EQU1##
Accordingly, high catalytic capacity can be achieved by increasing the specific activity of the enzyme (S.A.=umoles/min/mg enzyme) and/or extending enzyme half-life (t.sub.1/2)
The prior art has focused on the identification of a suitable organism and alcohol oxidase enzyme, and has particularly focused on yeasts (organisms which are common to the fermentation art), e.g., Kloeckera Sp. No. 2201 (Tani et al., Agr. Biol. Chem. 36, 76-83 [1972]) Candida Boidinii (Sahm and Wagner, European J. Biochem, 36, 250-256 [1973]), and Pischia pastoris. (Ellis, et al., Mol. Cell Biol. 5, 1111-1121 [1985]). Another such organism is Hansenula polymorpha. The first step in the methanol utilization mechanism of these organisms is the aerobic oxidation of methanol into formaldehyde and hydrogen peroxide. EQU CH.sub.3 OH+O.sub.2 .fwdarw.HCHO+H.sub.2 O.sub.2
In the in vivo system, the resulting hydrogen peroxide is rapidly decomposed by a catalase into oxygen and water. Levine and Cooney, Appl. Microbiol. 26(6), 982-990 (1973) isolated from soil a strain of Hansenula polymorpha, designated DL-1 (ATCC 26012) in which the methanol utilizing enzyme was thermotolerant (up to 50.degree. C. for free cells) and therefore compatible with conventional fermentation conditions. Van Dijken, et al., Arch. Microbiol. 111, 137 (1976) reported that this enzyme can be induced to a level of 20% of total soluble cell protein in Hansenula polymorpha.
Barratti, et al., Biotechnology and Bioengineering 20, 333-388 (1978), using cell-free extracts of Hansenula polymorpha DL-1, found that although conversion yields were excellent (98%), the methanol oxidizing enzyme had limited substrate tolerance (100 mM or about 0.04% methanol) and that at high concentrations of enzyme, dissolved oxygen became rate-limiting. Later, Couderc and Barratti, Biotechnology and Bioengineering 22, 1155-1173 (1980), developed techniques to increase the substrate tolerance of the enzyme in cells to 500 mM at the expense of conversion yield, and noted that the enzyme was inhibited at low levels of the product, H.sub.2 O.sub.2.
The inventors have found that enzyme from Hansenula polymorpha ATCC 34438 is relatively free from the potentially limiting factor of inactivation by feedstock concentration and inhibition by product concentration. Moreover, this strain of Hansenula, when grown under methanol limited conditions, could produce enzyme in a concentration of at least 20% of a cell-free extract, thus providing a relatively concentrated source of enzyme without extensive purification.
When reaction conditions were optimized within the conventional range, the inventors found that this alcohol oxidase was able to produce formaldehyde and hydrogen peroxide under conditions of higher product and feedstock concentrations, representing more than an order of magnitude improvement over the prior art.
It is well established that the properties of enzymes are affected by temperature. Although many proteins are stabilized somewhat by lowering the temperature below 25.degree. C., others exhibit cold lability. Bock, et al., TIBS 3, 100-103 (1978). Temperature changes often change the conformation of an enzyme, which in turn may affect enzyme properties in what is still a relatively unpredictable manner. See, e.g., Griep, et al., Biochemistry 25, 6688-94 (1986) (factor XII activation optimized at low temperatures); Isohashi, et al., Eur. J. Biochem. 142 (1984) (acetyl-CoA hydrolase inactivated at low temperature); Somero, J. Exp. Zool. 194, 175-88 (1975) (review of temperature effects on enzymes).
The present invention employs radical reaction conditions of low temperatures and high feedstock concentration to increase the catalytic capacity of certain alcohol oxidase enzymes from Hansenula and Pischia at least sixty-fold and encompasses the discovery that this surprising and unexpected result extends to analogous systems using alcohol oxidases from related organisms.
Accordingly, it is an object of the invention to provide a process for the enzymatic conversion of alcohol to aldehyde and hydrogen peroxide that increases the catalytic capacity of these enzymes to a commercially viable level.